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Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo.

ABSTRACT: Glucocorticoid-induced osteoporosis is a frequent complication of systemic glucocorticoid (GC) therapy and mainly characterized by suppressed osteoblast activity. Wnt16 derived from osteogenic cells is a key determinant of bone mass. Here, we assessed whether GC suppress bone formation via inhibiting Wnt16 expression. GC treatment with dexamethasone (DEX) decreased Wnt16 mRNA levels in murine bone marrow stromal cells (mBMSCs) time- and dose-dependently. Similarly, Wnt16 expression was also suppressed after DEX treatment in calvarial organ cultures. Consistently, mice receiving GC-containing slow-release prednisolone pellets showed lower skeletal Wnt16 mRNA levels and bone mineral density than placebo-treated mice. The suppression of Wnt16 by GCs was GC-receptor-dependent as co-treatment of mBMSCs with DEX and the GR antagonist RU-486 abrogated the GC-mediated suppression of Wnt16. Likewise, DEX failed to suppress Wnt16 expression in GR knockout-mBMSCs. In addition, Wnt16 mRNA levels were unaltered in bone tissue of GC-treated GR dimerization-defective GR ^dim mice, suggesting that GCs suppress Wnt16 via direct DNA-binding mechanisms. Consistently, DEX treatment reduced Wnt16 promoter activity in MC3T3-E1 cells. Finally, recombinant Wnt16 restored DEX-induced suppression of bone formation in mouse calvaria. Thus, this study identifies Wnt16 as a novel target of GC action in GC-induced suppression of bone formation.

SUBMITTER: Hildebrandt S

PROVIDER: S-EPMC5992207 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo.

Hildebrandt Susanne S Baschant Ulrike U Thiele Sylvia S Tuckermann Jan J Hofbauer Lorenz C LC Rauner Martina M

Scientific reports 20180607 1

Glucocorticoid-induced osteoporosis is a frequent complication of systemic glucocorticoid (GC) therapy and mainly characterized by suppressed osteoblast activity. Wnt16 derived from osteogenic cells is a key determinant of bone mass. Here, we assessed whether GC suppress bone formation via inhibiting Wnt16 expression. GC treatment with dexamethasone (DEX) decreased Wnt16 mRNA levels in murine bone marrow stromal cells (mBMSCs) time- and dose-dependently. Similarly, Wnt16 expression was also supp ...[more]

PMID: 29880826

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Project description:BackgroundPeriosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs.MethodsWe detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16.ResultsWnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity.ConclusionTogether, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

Project description:AIM: To investigate the mechanism of the bone-forming effects of intermittent parathyroid hormone (PTH) administration and to search for novel molecules of bone anabolism via the PTH signaling pathway. METHODS: Primary cultures of rat osteoblasts (ROBs) were divided into an intermittent PTH-treated group (Itm) and a control group (Ctr). Imitating the pharmacokinetics of intermittent PTH administration in vivo, the ROBs in the Itm group were exposed to PTH for 6 h in a 24-h incubation cycle, and the ROBs in the Ctr group were exposed to vehicle for the entire incubation cycle. The cells were collected at 6 h and 24 h of the final cycle, and the proteins in the Itm and Ctr groups were analyzed by two-dimensional electrophoresis (2-DE) coupled with peptide mass fingerprinting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to detect proteins that were differentially expressed. The proteins with the most significant changes in vitro were validated by immunohistochemistry (IHC) in a rat model. RESULTS: The proteomics analysis indicated that a total of 26 proteins were up- or down-regulated in the Itm group compared with the Ctr group at 6 h and 24 h; among these, 15 proteins were successfully identified. These proteins mainly belong to the cytoskeleton and molecular chaperone protein families, and most of these have anti-apoptotic effects in various cells. Rho GDP-dissociation inhibitor alpha (RhoGDIalpha) and vimentin were the most significantly changed proteins. Further studies by IHC showed that the expression of RhoGDIalpha in ROBs was significantly higher in PTH-treated sham-operated rats than in vehicle-treated sham-operated rats, but the difference was not significant between PTH-treated and vehicle-treated OVX rats. Vimentin expression was not changed in either PTH-treated sham-operated rats or PTH-treated OVX rats. CONCLUSION: Our research suggests that intermittent PTH treatment induces changes in expression of many proteins in ROBs in vitro, and it results in RhoGDIalpha up-regulation in ROBs both in vitro and in vivo when estrogen is present. This up-regulation of RhoGDIalpha may be one of the mechanisms underlying the synergistic bone-forming effect of PTH and estrogen.Acta Pharmacologica Sinica (2009) 30: 1001-1007; doi: 10.1038/aps.2009.60.

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Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo.

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Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo.

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